The Study For The Anti-leukemia Effects Of Resveratrol And Underlying Mechanism

Part I Inhibitory Effect of Resveratrol on Proliferation ofLeukemic CellsObjective: To investigate the effect of leukemic cellsproliferation, cell cycle and apoptosis by resveratrol and make the theoretics and experiment basis for reveratrol as an antileukemia drug.Methods: Human K562 leukemia cells were cultured in RMPI1640 mediumsupplemented with 10% fetal bovine serum in 37℃, 5%CO₂ humid incubator. The medium was changed every other day. In all experiments, the viability of cells was over 98%. Peripheral blood mononuclear cells (PBMNC) obtained from healthy individuals were separated by Ficoll solution and suspended in above mentioned medium. It was detected the inhibitory reaction of K562 cell and NPBMNC to Res on various concentration. Annein-V-FITC/PI double staining flow analysis of cytometre skills(FACS) was used to detect the effect of Res on cell cycles and apoptosis in K562 cells.Results: (1) There was significant difference in the inhibition rate between the control group and Res group, the inhibition was in a time and dose dependent manner. In a special range of concentration, Ras had no remarkable effect on PBMNC, which demonstrated that Res could selectively inhibit K562 cell proliferation. (2) Increased concentration of Ras can swith the cell cycles of K562 cell, induced cell cycle arrest at S phase, decreased the proportion of cells in G₂/M phase. (3)Res can induce K562 cells apoptosis in a dose dependant manner.Conclusion: Res can regulate cell cycles of K562 cells and indueapoptosis to inhibit the proliferation of K562 cells. Res can selectively inhibit leukemic cells proliferation and has no cytotoxic effect on PBMNC.Part II the Effect and significance ofResveratrol on leukemic cells Renin-Angiotensin SystemObjectives: To study the different level of renin in leukemiapatients group and control group, the growth promoting effect of angiotensin II on leukemic cell in vitro and the effect of Res on the level of Ang II . To explore the role of RAS in development of leukemia and mechanism of Res against leukemia.Methods: Concentrations of renin in BM and peripheral blood samples from 30 patients with acute leukemia or chronic leukemia were detected by radioimmunoassay, 7 thrombocytopenic purpura patients as controls. Human K562 leukemia cells were cultured in RMPI1640 medium supplemented with 10% fetal bovine serum in 37℃, 5%CO₂ humid incubator. The medium was changed every other day. In all experiments, the viability of cells was over 98%. AngII act on K562 cell with various concentrations, the reaction of growth was detected by MTT method. 100 and 200 μmol ·L^-1 Res treat K562 cell for 48 hours, the concentrations of AngII in cultural medium assayed by radioimmunoassay.Results: (1)Concentrations of the BM renin in acute leukemia groupwere higher than in control group, which was statistically significant difference (P<0.05). But concentrations of the BM renin in chronic leukemia group were no significantly higher than the control group(P=0. 562). In eight acute leukemia patients, the renin concentration was found to be significantly higher in the BM than in the peripheral blood (P=0.043). (2) AngII had the growth promoting effect on K562 cells with a dose and time dependent manner within a special range. (3) Concentration of Ang II in K562 cell cultural medium significantly decreased by Res treated for 48h.Conclusion: There was abnormal level of renin in the leukemia patient bone marrow; AngII had promoted K562 cells proliferation; there exists AngII autocrine in leukemic K562 cells. Res can inhibit AngII production in a dose dependent manner. It was supposed that Res inhibited the proliferation of leukemia cell by inhibiting AngII production due to its angiotensin converting enzyme inhibitor activity. PartIII Inhibitory Effect and mechanism of Resveratrol on AngiogenesisObjective: To study effect of Res on endothelial cell prolifertion andRes on leukemic cell vascular endothelial growth factor secretion. To explore effect of Res on leukemia angiogenesis.Methods: human umbilical vein endothelial cell(HUVEC) culture in vitroand leukemic U937 cells as research objects. Separated HUVEC were grown in 25cm² plastic culture flask coated with 2% gelation until cells were grown to confluence in monolayer. These cells identified to be endothelial cell by VIII factor SABC method. 3～6 era cell was as research object. Various concentration Res affect HUVEC capillary formation by research the vascular tube formation in vitro. U937 culture in above metioned method, ELISA method was used to analyse the effect of Res on VEGF level in cultural medium.Results: (1)Normal HUVECs formed integrated tube-like formation onmatrigel. Ras can inhibit formation of vessel-like structures induced by matrigel with a dose dependent manner. (2)There was statistically significant decrease in VEGF concentration, when Res treated on U937 cells for 24h or 48h, compared with blank control group. These showed that Res inhibited U937 VEGF secretion related with Res concentration.Conclusion: Res can significantly inhibit HUVECs microvasculartube-like formation in vitro, which showed Res had an antiangiogenesis activity. There was VEGF autocrine in Leukemic U937 cell, Res can significantly inhibit U937 cell VEGF secretion. Res inhibit angiogenesis was portion mechanism of Res antileukemia.